Figure 1.
Fludarabine is converted in serum to a dephosphorylated form by a 5′nucleotidase [37]. PNP attached to the cell surface via annexin V and phosphatidylserine binding then cleaves the ribose-1-phosphate group, resulting in 2-fluoroadenine [11], [12]. The freely diffusible molecule enters the cell and inhibits protein, RNA, and DNA synthesis [13]. Nucelotide-specific membrane transport carriers transport the dephosphorylated form across the cell membrane, where it is then phosphorylated into a cytotoxic triphosphate [3], [37].
Figure 2.
PNP-AV, PNP, AV protein models.
Images generated with The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC with data files obtained through the Protein Data Bank Europe. (a) Image of human annexin V generated from the crystal structure [38]. (b) Image of Escherichia coli PNP [39]. c Mesh image of a model of PNP-AV hexamer.
Figure 3.
MCF-7 dissociation constant binding data.
Specific binding (▴) was determined by subtracting total binding (•) in calcium supplemented medium from nonspecific binding (▪) in calcium deficient medium. Binding was quantified with biotinylated protein and HRP-conjugated streptavidin, developed with OPD. Data are presented as mean ± SE (n = 3).
Table 1.
Dissociation constant (Kd) of PNP-AV binding to cells.
Figure 4.
Bound fusion protein over a 3-7 (♦), MDA-MB-231 (▴), and HAAE-1 (▪) cell lines. Data are presented as mean ± SE (n = 3).
Figure 5.
PNP-AV membrane binding visualization.
Confocal microscopy of MCF-7 cells confirms the presence of externally bound biotinylated PNP-AV, with streptavidin-conjugated Alexa Fluor 488 in green (a, e), red CellMask stain of the plasma membrane (b, f), and blue Hoechst 33258 dye staining of nucleic acids (c, g). Images a-c show the color channels of an x-y confocal image with the composite shown in d. Images e-g show the color channels of an x-z cross-section of cells with the composite shown in h and the coverslip indicated by the white in each image.
Figure 6.
In vitro cytotoxicity of enzyme prodrug treatment.
The effects of fludarabine, 2-fluoroadenine, and fludarabine converted to 2-fluoroadenine by PNP are shown for (a) non-confluent HAAE-1 cells, (b) MCF-7 cells, and (c) MDA-MB-231 cells. Groups that received PNP-AV were treated on days 0 and 3 of the study. Fludarabine and 2-fluoroadenine were administered daily. Viability was determined by the Alamar Blue assay on days 2, 4, and 6 (black, gray, and white bars, respectively), and each sample was represented as a percentage of untreated control on each day. Statistical analysis was performed with a one-way ANOVA test with data presented as mean ± SE (n = 3). Statistical significance vs. untreated control on the same day is denoted by *(p<0.001).